Covalently closed, circular DNA in kappa endosymbionts of<i>Paramecium</i>

Dilts, Judith A.

doi:10.1017/s0016672300016360

Cited by 29 publications

(18 citation statements)

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“…Indirect evidence indicates that the genetic determinants for R-body and toxin synthesis are extrachromosomal (2,9,10). In three of the four species of Caedibacter, bacteriophage-like structures are associated with R-body and toxin production (2,9).…”

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“…In three of the four species of Caedibacter, bacteriophage-like structures are associated with R-body and toxin production (2,9). In the fourth species, Caedibacter taeniospiralis, R-body and toxin production are associated with plasmids (10) Plasmid DNA Preparation. C. taeniospiralis 47 was purified from lysates of host paramecia by chromatography on ECTEOLA columns (7).…”

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confidence: 99%

“…C. taeniospiralis 47 was purified from lysates of host paramecia by chromatography on ECTEOLA columns (7). Plasmid DNA (pKAP47) was separated by using ethidium bromide/cesium chloride gradients (10). Plasmid DNA was obtained from E. coli by using the cleared lysate method of Clewell and Helinski (12).…”

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Cloning and expression of DNA sequences associated with the killer trait of Paramecium tetraurelia stock 47.

Quackenbush¹,

Burbach²

1983

Proc. Natl. Acad. Sci. U.S.A.

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We have presented direct evidence that at least one of the traits associated with killing of paramecia by kappa particles is determined by an extrachromosomal genetic element. Plasmid DNA was isolated from Caedibacter taeniospiralis 47 (commonly known as 47 kappa), which is an obligate cytoplasmic endosymbiont of Paramecium tetraurelia. Fragments of pKAP47 DNA generated by Pst I digestion were inserted into pBR328 and then introduced into Escherichia coli 294 by transformation. Clones carrying recombinant plasmids were screened for toxicity toward sensitive strains of paramecia or for the ability to produce R bodies. None of the clones appeared to be toxic. However, three clones were found to have the ability to produce R bodies, which are proteinaceous ribbons (10-20 ,um long, 0.5 jam wide, and 13 nm thick) rolled up inside the cell to form a hollow cylinder about 0.5 ,um in diameter and 0.5 jzm long. Each of these clones carry plasmids that contain the Pst I B fragment from pKAP47. Subclones of one of the recombinant plasmids, pBQ51, were constructed to determine the approximate location of DNA sequences necessary for R-body synthesis. The left-hand boundary of the required sequences was found to occur within a 600-base-pair region, and the location ofthe right-hand boundary was determined to occur within a 700-base-pair region. The minimum and maximum sizes ofsequences required for R-body synthesis are between 1,300 and 2,600 base pairs.Killer traits in paramecium are genetically determined by a variety of bacterial endosymbionts that live in the cytoplasm of killer paramecia (1, 2). The most notable group ofthese bacteria are kappa particles, which comprise the genus Caedibacter. Killer paramecia release these endosymbionts, some of which carry toxin, at a slow rate into the environment. These endosymbiont-bearing paramecia are killers because uninfected paramecia that are sensitive to the toxin may suffer lethal consequences when toxic forms of endosymbionts are ingested.All members of the genus Caedibacter, in addition to being cytoplasmic endosymbionts that can confer a killer trait on their host paramecia, have two distinct morphological forms (3). The predominant form (about 95% of most populations) does not exhibit any unusual morphological features. The second form, which accounts for about 5% of the individuals in most populations, contains a large inclusion body (Fig. 1A) known as an R body. Inside the cell, R bodies appear as hollow cylinders approximately 0.5 ,um long and 0.5 Am in diameter. An R body (Fig. 1B) is actually a long (8-20 ,um) proteinaceous ribbon, about 0.5 ,um wide and 13 nm thick, that is rolled up inside the bacterial cell to form a hollow cylinder (4). Several investigators have shown that it is the R-body-containing forms of the endosymbionts that are toxic and that they are the progeny ofcells that do not contain R bodies (5-8).Indirect evidence indicates that the genetic determinants for R-body and toxin synthesis are extrachromosomal (2, 9, 10). In three of the fo...

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Cloning and expression of DNA sequences associated with the killer trait of Paramecium tetraurelia stock 47.

Quackenbush¹,

Burbach²

1983

Proc. Natl. Acad. Sci. U.S.A.

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“…Dilts (2) was the first to demonstrate the presence of extrachromosomal DNA in bacteriumlike endosymbionts. She showed that Caedibacter taeniospiralis (kappa), a bacterial endosymbiont isolated from the fresh water ciliate Paramecium tetraurelia stock 51, contained covalently closed circular DNA molecules of contour length 13.75 ,um, corresponding to a molecular weight of 2.8 x 107, and suggested that this DNA might be the genetic determinant for the formation of R bodies and the helical phagelike structures present in the symbiont.…”

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Association of transformation of xenosomes from nonkiller to killer with extrachromosomal DNA

Soldo¹,

Brickson²,

Castiglione³

et al. 1986

J Bacteriol

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Extrachromosomal DNA in the form of covalently closed circular DNA molecules was isolated from killer and nonkiller xenosomes, bacterial endosymbionts of the marine protozoan Parauronema acutum. Restriction endonuclease digests of these molecules derived from 12 isolates revealed consistent, readily identifiable, differences in the pattern of fragments of the killer as compared with those present in the nonkiller. Transformation of the nonkiller to killer by infection is also accompanied by a change from the nonkiller to killer pattern. Based on analysis of fragments resulting from restriction endonuclease digests, two circular duplex DNA molecules, each 63 kilobase pairs (kbp) in length, were identified in the 263-20 nonkiller stock and mapped. The maps revealed that each possesses a single BamHI site and multiple Bgll, BstIIE, PstI, and Sall sites. A distinguishing feature of these maps is that the two molecules share a region about 17 kbp in length in which multiple restriction sites are in register with each other. Allowing for a 0.5-kbp insertion or deletion and the introduction or removal of only a few restriction sites, an additional stretch extending approximately 31 kbp beyond this sequence could also be considered to be homologous. The structure of the killer plasmid appears to be more complex, and we have been unable, as yet, to construct physical maps for this DNA. We postulate that the killer plasmid DNA is composed of three, perhaps four, circular 63-kbp duplexes, at least one which contains a single BamHI site and another which contains two BamHI sites. The remaining molecules may represent copies of either or both of the other two, modified to contain additional restriction sites. Transformation from the nonkiller to the killer is visualized as the insertion of restriction sites at various points along parent nonkiller plasmid DNA molecules. The mechanism by which these sites are introduced is unknown.Several stocks of the marine protozoan Paraiuronema acutum contain intracytoplasmic symbiotic bacteria which grow and divide in synchronism with the host (13). The symbionts, which we have termed xenosomes, sensu strictu, are obligate parasites and cannot be cultured outside the protozoan. Xenosomes released from the cytoplasm by mechanical rupture of the host cell are capable of infecting homologous and heterologous strains of P. acutuem as well as one other marine protozoan, Miamiensis aliidus Ma. Xenosomes from some stocks (killers) are capable of killing certain marine protozoa of the genus Uronema as evidenced by exposure of these organisms to breis of symbiont-bearing P. acutum. We show here that xenosomes from other stocks of P. acutum (nonkillers) do not have this capacity. Significantly, P. acutum is insensitive to the toxin produced by the killer xenosomes. In an effort to determine the mechanism responsible for the killer effect and to establish its molecular basis, we previously examined chromosomal DNA derived from killer and nonkiller xenosomes by DNA-DNA hybridization (9). Whereas a h...

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“…R bodies are long (approximately 10 ,um), proteinaceous ribbons about 0.5 ,um wide and 13 nm thick, which are rolled up inside the bacterial cell, forming hollow cylinders (1). All strains of C. taeniospiralis carry plasmids (2,7,8). The functions of these plasmids are, for the most part, unknown.…”

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Transposonlike elements in Caedibacter taeniospiralis

Quackenbush¹,

Dilts²,

Cox³

1986

J Bacteriol

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We report that the 1.5-and 7.5-kilobase-pair (kbp) transposonlike sequences present in the R-body-coding plasmids of Caedibacter taeniospiralis share homology. The R-body-coding plasmids of two new strains of C. taeniospiralis, derived from strains 169 and A30, carry the 7.5-and 1.5-kbp elements, respectively, inserted at new positions. Sequences homologous to the 7.5-kbp sequence from C. taeniospiralis 47 were detected in the chromosomes of three other strains of C. taeniospiralis.All members of the bacterial species Caedibacter taeniospiralis are obligate cytoplasmic endosymbionts of the freshwater ciliate Paramecium tetraurelia. These bacteria, commonly known as hump killer kappa particles, confer the hump killer trait on their protozoan hosts. In addition, C. taeniospiralis is notable in its ability to synthesize a large inclusion body known as an R body. R bodies are long (approximately 10 ,um), proteinaceous ribbons about 0.5 ,um wide and 13 nm thick, which are rolled up inside the bacterial cell, forming hollow cylinders (1). All strains of C. taeniospiralis carry plasmids (2,7,8). The functions of these plasmids are, for the most part, unknown. However, one function (R-body production) has been demonstrated (9), and another (the killer trait) has been suggested (2).Restriction endonuclease analysis has shown that the R-body-coding plasmids of six strains of C. taeniospiralis have very similar physical maps (8). In every case, the differences observed among these plasmids can be accounted for by single insertion events involving DNA sequences that are about 1.5 or 7.5 kilobase pairs (kbp) in length. In this communication, we report that these inserted sequences share homology with each other and that they appear to be transposable genetic elements.The strains of C. taeniospiralis used in this study are listed in Table 1. Plasmid DNA was purified from these strains with ethidium bromide-cesium chloride gradients as previously described (10). Both upper and lower bands were collected from gradients. Conditions for restriction endonuclease digestions and agarose gel electrophoresis were as previously described (10). Agarose gels were stained for approximately 5 min in ethidium bromide (10 jig/ml) and photographed. To obtain Southern blots (14) of agarose gels, we denatured restriction endonuclease-generated DNA fragments in situ and then transferred them to Gene Screen membranes (New England Nuclear Corp., Boston, Mass.) per the manufacturer's instructions. The recombinant plasmid pBQ54 (9) was used as a probe in Southern blot hybridizations. Probe DNA was radiolabeled by nick translation with 32P-dCTP (ICN Pharmaceuticals, Inc., Irvine, Calif.) as previously described (6 radiolabeled pBQ54 were carried out at 60°C by the method described by Maniatis et al. (5). Physical maps of pKAP30-1 and pKAP169-1 were determined by restriction endonuclease analysis as previously described (10), in which DNA fragment profiles generated by single and double restriction endonuclease digestions were compared. Restriction en...

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Covalently closed, circular DNA in kappa endosymbionts ofParamecium

Cited by 29 publications

References 18 publications

Cloning and expression of DNA sequences associated with the killer trait of Paramecium tetraurelia stock 47.

Cloning and expression of DNA sequences associated with the killer trait of Paramecium tetraurelia stock 47.

Association of transformation of xenosomes from nonkiller to killer with extrachromosomal DNA

Transposonlike elements in Caedibacter taeniospiralis

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